** [https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=603176 Applied Biosystems Catalog Web Page]. Originally an Ambion product. Ambion was acquired by Applied Biosystems and the kit can only be purchased through them.

+

** [https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=603176 Applied Biosystems Catalog Web Page]. Originally an Ambion product. Ambion was acquired by Applied Biosystems which has now merged with Invitrogen and is called "Life Technologies" and the kit can only be purchased through them.

** Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.

+

* 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene

-

** Can be re-used

+

* 200 mL needed to fill Coplin jar

+

* Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.

+

* Can be re-used (but not indefinitely)

+

* NOTE: PEG 8000 does '''NOT''' work in this recipe. It will not dissolve in the acetone. I have not tried MW cut-offs between 2000 and 8000, though. ''&mdash; [[User:Kam D. Dahlquist|Kam D. Dahlquist]] 12:36, 22 July 2010 (EDT)''

* Once the Cy3 and Cy5 dye packages have been opened, all procedures must be carried out in the dark.

* Once the Cy3 and Cy5 dye packages have been opened, all procedures must be carried out in the dark.

-

* Generally, we perform up to 10 reactions at one time.

+

* Generally, we perform up to 10 reactions (5 slides) at one time.

* Make sure to perform multiple reactions for the reference sample so that there is enough aRNA for each of the chips.

* Make sure to perform multiple reactions for the reference sample so that there is enough aRNA for each of the chips.

* If using kit for the first time, add the appropriate amount of 100% ethanol to the Wash Buffer.

* If using kit for the first time, add the appropriate amount of 100% ethanol to the Wash Buffer.

===Day 1===

===Day 1===

+

+

'''''Note that Ambion/Applied Biosystems/Life Technologies revised the protocol for the AM1753 Amino Allyl MessageAmp II aRNA Amplification Kit and we started using the revised protocol on 12/9/10.'''''

#* Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.

#* Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.

Line 111:

Line 115:

#* Use 16°C water bath in the cold room for step D2.

#* Use 16°C water bath in the cold room for step D2.

#* Potential stopping point for Day 1. Freeze samples at -20°C. Protocol says it is better to go on to cDNA purification before freezing, though.

#* Potential stopping point for Day 1. Freeze samples at -20°C. Protocol says it is better to go on to cDNA purification before freezing, though.

-

# '''cDNA Purification:''' Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 50°C in a water bath (the protocol says to heat the entire bottle, but this is unwieldy).

+

# '''cDNA Purification:''' Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 55°C in a water bath (the protocol says to heat the entire bottle, but this is unwieldy).

-

# Perform steps E1-E4.

+

# Perform steps E1-E5.

# Stopping point for Day 1. Freeze samples at -20°C.

# Stopping point for Day 1. Freeze samples at -20°C.

Line 123:

Line 127:

# Perform steps G1-G5.

# Perform steps G1-G5.

#* Do not vortex and do not centrifuge in steps G1 and G2 and proceed as quickly as possible to step G3, otherwise the aRNA will start precipitating and you will lose it.

#* Do not vortex and do not centrifuge in steps G1 and G2 and proceed as quickly as possible to step G3, otherwise the aRNA will start precipitating and you will lose it.

+

#* We have also noticed a degradation in quality of the spin columns supplied by Ambion (now Invitrogen) in this kit. The filters do not stay seated in the column and the column material seems to come down into the elution tube. To remedy this, we have begun to spin the columns at half the speed and for twice as long a time as called for in the protocol. ''&mdash; [[User:Kam D. Dahlquist|Kam D. Dahlquist]] 12:55, 22 July 2010 (EDT)''

# Stopping point for Day 2. Freeze samples at -80°C.

# Stopping point for Day 2. Freeze samples at -80°C.

Line 130:

Line 135:

# Make a 1:50 dilution of each aRNA sample by diluting 2 μL of sample into 98 μL of TE Buffer.

# Make a 1:50 dilution of each aRNA sample by diluting 2 μL of sample into 98 μL of TE Buffer.

# Before beginning the procedure, pipet enough nuclease-free water into a fresh tube so that you have 20 μL X number of tubes, plus a little extra. Pre-heat to 50-60°C. The water should heat for at least 10 minutes.

+

# Before beginning the procedure, pipet enough nuclease-free water into a fresh tube so that you have 30 μL X number of tubes, plus a little extra. Pre-heat to 55°C. The water should heat for at least 10 minutes.

-

# Add 150 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.

+

# Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.

# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''

# Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. '''''Do NOT vortex and do NOT centrifuge.'''''

-

# Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. Spin 1 minute at 10,000 X g. Discard flow-through.

+

# Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. Spin <s>1 minute at 10,000 X g</s> 2 minutes at 5,000 X g. Discard flow-through.

#* ''Note that we started having issues with the spin columns when Ambion/Applied Biosystems got purchased by Invitrogen and they started supplying Invitrogen columns in the kit. The resin would come down into the collection tube. We now spin for 2 minutes at 5,000 X g instead.''

#* Note: the manufacturer's protocol calls for eluting twice with ''10 μL'' of the nuclease-free water, not 15 μL. However, we have observed that 5-8 μL of the total elution volume is retained on the column. By increasing the volumn slightly for each elution step we have increased our yield of labeled aRNA without diluting it too much. ''&mdash; [[User:Kam D. Dahlquist|Kam D. Dahlquist]] 12:47, 22 July 2010 (EDT)''

-

# Repeat the elution with another 10 μL of nuclease free water pre-heated to 50-60°C.

# Place the cover slip with the smooth side up on a support (a rack for 15 mL test tubes works great). Have the microarray slide ready with the hybridization chamber. Pipet the hybridization solution in a line on the cover slip, avoiding the introduction of air bubbles. Holding the microarray slide only by the edges (do not touch the front or back of the slide), pick up the cover slip with the DNA side of the slide down (the label side is the DNA side) and quickly invert the slide and place in the chamber with the DNA side and cover slip up.

+

# Pipet 11 μL of nuclease-free water into the wells in the hybridization chamber on either side of the slide. Place the cover on the chamber and lock the clips in place.

+

# Working quickly and keeping the hybridization chamber horizontal, place the chamber at the bottom of the 37°C water bath. Incubate for 15-16 hours.

+

+

=== Day 5 ===

+

+

'''Post-hybridization washing and scanning'''

+

+

# Remove the hybridization chamber from the water bath and dry the outside with a large kim wipe. Pull the clamps off and wipe again before opening the chamber. Lift the slide from the barcode end using forceps. Remove the coverslip by quickly, but gently dipping the slide in a 50 mL conical tube filled with room temperature 1X SSC. Let the coverslip slide off gently; hold the slide at the barcode end with forceps. Once the cover slip comes off, transfer the slide to another conical tube filled with 50°C 1X SSC/0.1% SDS. Incubate for 15 minutes in the 50°C waterbath.

+

# Transfer slide quickly to a second tube filled with 50°C 1X SSC/0.1% SDS. Do not let the slide dry out while transferring. Incubate for 15 minutes in the 50°C waterbath.

+

# Transfer slide quickly to a third tube filled with 50°C 1X SSC/0.1% SDS. Do not let the slide dry out while transferring. Incubate for 15 minutes in the 50°C waterbath.

+

# After the three washes are complete, rinse the slide in a tube filled with room temperature 1X SSC by plunging up and down 4-6 times.

+

# Rinse the slide in a tube filled with room temperature 0.1X SSC by plunging up and down 4-6 times. When you pull the slide out the last time, pull it out very slowly so that water droplets do not form on the surface of the slide. Then carefully blot the edges of the slide dry on a folded kim wipe, being careful not to touch the front or back surface of the slide. Use the compressed air duster to blow off any water droplets that remain. You are not blowing the water dry, but chasing it to the edge of the slide so that it can be blotted by the kim wipe.

+

# Carry the dry slide in a slide box covered in foil to the fume hood in the Keck Lab. Dip the slide into the coplin jar filled with 2% PEG (2000) in 1:1 acetone:toluene. Be careful not to dip the label into the solvent. Work quickly. Allow the slide to dry for about 1 minute before putting it back in the box and bringing it back into the Dahlquist lab for scanning. Keep the slide in the dark until scanning.

+

+

'''Scanning with the Axon GenePix 4000B Scanner'''

+

+

# Make sure that the computer is OFF before turning on the scanner.

+

# Remove the dust cover and turn on the scanner using the switch on the black box in the back of the scanner.

+

# Turn on the computer and login to the username “scanner”. Ask Dr. Dahlquist for the password.

+

# Let the scanner warm up for 15 minutes before scanning.

+

# See the pictures on the cabinet above the scanner for how to insert the slide. Be gentle!

+

#* The DNA side of the chip should be down and the barcode should be facing towards the front of the instrument.

+

# Open the Hardware Settings window (click the icon on the right-hand side that looks like the scanner with a key hanging off of it.)

+

# Make sure that the pixel size is set to 10, lines to average is set to 2, and focus position is set to 0. Set the PMT Gain for the 635 wavelength to 650 and the PMT Gain for the 532 wavelength to 550. The Power should be at 100% for both.

+

# Perform a Preview Scan by clicking the icon with the double arrows (looks like a "fastforward" icon).

+

#* While it is scanning, click on the histogram tab to see the Count Ratio of red to green signal. It should be within 10% of 1.00 (0.90 to 1.10). You can adjust the PMT Gain settings in the Hardware Settings window to adjust this ratio. The histogram tab will show you the data for the area of the image that is selected in the main Image window, so if you change the settings, you need to zoom to the area in which that setting applies.

+

#* Allow the Preview Scan to complete, so that the instrument will read the barcode. If it does not register the bar code, double-click on the barcode area at the bottom of the screen to enter it manually.

+

# Perform a Data Scan by clicking on the single arrow (second icon down on the right-hand side of the window). It will take ~10 minutes to complete the scanning of the slide.

+

# Save the data by clicking on the floppy/folder icon on the right hand side and choosing "Save Images" from the menu that appears.

+

#* In the dialog box that appears, make sure that the images are being saved as "Single-image tiffs". Use the barcode and date as part of the file names. Create a folder for today's date (yyyymmdd) in the C:\MicroarrayData folder to save the files.

+

# When you are finished scanning, turn off the scanner so that we do not wear out the laser and replace the dust cover.

+

# Go to the [[Dahlquist:GenePix_Pro_Software_Protocol | GenePix Pro Software Protocol]] for gridding the image and generating the data.

==Notes==

==Notes==

-

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

+

Please feel free to post comments, questions, or improvements to this protocol on the [[Talk:{{PAGENAME}}|talk page]]. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

-

#List troubleshooting tips here.

+

-

#You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.

+

-

#Anecdotal observations that might be of use to others can also be posted here.

+

-

Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.

+

<!-- ==References==

-

+

-

==References==

+

'''Relevant papers and books'''

'''Relevant papers and books'''

If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information.

If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information.

Reagents

Applied Biosystems Catalog Web Page. Originally an Ambion product. Ambion was acquired by Applied Biosystems which has now merged with Invitrogen and is called "Life Technologies" and the kit can only be purchased through them.

Nuclease-free water is provided in the Amino Allyl MessageAmp™ II aRNA Amplification Kit and is sufficient for the μL quantities called for throughout the protocol; additional water can be purchased from Applied Biosystems/Ambion, but it is expensive.

DEPC is an oxidizer and should be fully removed from water used with DNA microarrays because the Cy5 dye is prone to degradation by oxidation.

For larger quantities of nuclease-free water (non-DEPC treated) that are needed to dilute 20X SSC for washes, for example, I use Water, ASTM Type II, non-sterile, Reagent Grade, ACS (VWR Catalog #RC91505) that can be purchased in 10 L or 20 L cubes. This water was recommended by Genisphere in the DyeSaver 2 protocol as being validated for use with microarrays and not containing components that will oxidize Cy5; Kam D. Dahlquist 20:24, 15 August 2008 (EDT))

NOTE: they have discontinued printing these chips as of 7/31/10. — Kam D. Dahlquist 12:38, 22 July 2010 (EDT)

Slide Coating Solution

"Home-brew" version of Genisphere DyeSaver 2

2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene

200 mL needed to fill Coplin jar

Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.

Can be re-used (but not indefinitely)

NOTE: PEG 8000 does NOT work in this recipe. It will not dissolve in the acetone. I have not tried MW cut-offs between 2000 and 8000, though. — Kam D. Dahlquist 12:36, 22 July 2010 (EDT)

Procedure

General Notes

Use RNase-free reagents and supplies and maintain good RNase-free technique throughout.

At the beginning of each day's work, clean the bench top and pipetman shafts with RNaseZap.

Keep all samples on ice unless specifically noted otherwise.

Once the Cy3 and Cy5 dye packages have been opened, all procedures must be carried out in the dark.

Generally, we perform up to 10 reactions (5 slides) at one time.

Make sure to perform multiple reactions for the reference sample so that there is enough aRNA for each of the chips.

If using kit for the first time, add the appropriate amount of 100% ethanol to the Wash Buffer.

Day 1

Note that Ambion/Applied Biosystems/Life Technologies revised the protocol for the AM1753 Amino Allyl MessageAmp II aRNA Amplification Kit and we started using the revised protocol on 12/9/10.

Dry 1 μg of yeast total RNA to less than 10 μL in a SpeedVac.

1 μg is the largest amount recommended for the kit.

RNA can be dried ahead of time and stored at -80°C.

Reverse Transcription to Synthesize First Strand of cDNA: follow steps C1-C3 in protocol.

Use 42°C water bath for step C4.

Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.

Second Strand cDNA synthesis: perform steps D1-D2.

Use 16°C water bath in the cold room for step D2.

Potential stopping point for Day 1. Freeze samples at -20°C. Protocol says it is better to go on to cDNA purification before freezing, though.

cDNA Purification: Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 55°C in a water bath (the protocol says to heat the entire bottle, but this is unwieldy).

Day 2

Perform step F2 in 37°C water bath for six hours. The protocol says to incubate for 4-14 hours. However, it is important to incubate the same amount of time for each experiment to reduce variability.

Potential stopping point for Day 2. Freeze samples at -80°C.

aRNA Purification: Aliquot nuclease-free water needed for elution step (G5) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 50°C in a water bath.

Perform steps G1-G5.

Do not vortex and do not centrifuge in steps G1 and G2 and proceed as quickly as possible to step G3, otherwise the aRNA will start precipitating and you will lose it.

We have also noticed a degradation in quality of the spin columns supplied by Ambion (now Invitrogen) in this kit. The filters do not stay seated in the column and the column material seems to come down into the elution tube. To remedy this, we have begun to spin the columns at half the speed and for twice as long a time as called for in the protocol. — Kam D. Dahlquist 12:55, 22 July 2010 (EDT)

Amplified aRNA should appear as a smear from 250 to 5000 nt. The average size of amino allyl aRNA should be approximately 1400 nt.

Mix together aRNA samples if multiple reactions were performed for the same total RNA sample and each passes quality control. Re-read absorbance values and re-calculate concentration.

Calculate the volume required for 20 μg of aRNA and aliquot into a fresh 1.5 mL microcentrifuge for each sample (if you need multiple reference samples, make sure to aliquot enough to hybe all of your chips). Store all samples at -80°C. Stopping point for day 3.

Day 4

Indirect Labeling by Dye-Coupling

NOTE: this protocol uses the Applied Biosystems (formerly Ambion) Amino Ally MessageAmp II aRNA Amplification kit and GE (formerly Amersham) CyDye single reaction dye pack of the activated Cy3 and Cy5 dyes. The following steps are performed in the dark and are taken from the Ambion protocol.

Place 20 μg of amino allyl aRNA in a nuclease-free microfuge tube, and vacuum dry to completion on the low setting (takes 30-60 minutes, depending on volume). After 30 minutes, check every 5-10 minutes and remove sample as soon as it is dry; do not overdry.

Resuspend each CyDye pack in 11 μL of DMSO [and keep in the dark at room temperature for up to 1 hour until ready to use it.] You will need one Cy3 and one Cy5 pack per microarray.

It is helpful to flash spin the dried dye before adding the DMSO to make sure it is at the bottom of the tube.

To tube containing the aRNA:Coupling Buffer mixture, add 11 µL of dye to each tube, mix thoroughly and flash spin.

NOTE: Make sure you know which dye to add to which tube!

Incubate at room temperature in dark for 30 minutes (Wrap the rack with foil and place the rack in a drawer).

To quench the reaction, add 4.5 μL of 4 M hydroxylamine and mix well. Incubate at room temperature in the dark for 15 minutes.

Add 5.5 μL of nuclease-free water to bring each sample to 30 μL.

Removing Uncoupled Dye Material

Before beginning the procedure, pipet enough nuclease-free water into a fresh tube so that you have 30 μL X number of tubes, plus a little extra. Pre-heat to 55°C. The water should heat for at least 10 minutes.

Add 105 μL of aRNA Binding Buffer to each labeled aRNA sample--proceed to the next step immediately.

Add 75 μL of 100% ethanol to each sample, and mix by pipetting the mixture up and down 3 times. Do NOT vortex and do NOT centrifuge.

Place aRNA filter cartridges into the appropriate number of collection tubes and pipet each sample from the previous step onto the appropriate column. Spin 1 minute at 10,000 X g 2 minutes at 5,000 X g. Discard flow-through.

Note that we started having issues with the spin columns when Ambion/Applied Biosystems got purchased by Invitrogen and they started supplying Invitrogen columns in the kit. The resin would come down into the collection tube. We now spin for 2 minutes at 5,000 X g instead.

Transfer filter cartridge into a fresh collection tube, being careful not to contaminate the new tube with any of the flow through from the previous step.

To the center of the filter, add 15 μL of the nuclease-free water that you pre-heated to 55°C in step 1.

Note: the manufacturer's protocol calls for eluting twice with 10 μL of the nuclease-free water, not 15 μL. However, we have observed that 5-8 μL of the total elution volume is retained on the column. By increasing the volumn slightly for each elution step we have increased our yield of labeled aRNA without diluting it too much. — Kam D. Dahlquist 12:47, 22 July 2010 (EDT)

Place the cover slip with the smooth side up on a support (a rack for 15 mL test tubes works great). Have the microarray slide ready with the hybridization chamber. Pipet the hybridization solution in a line on the cover slip, avoiding the introduction of air bubbles. Holding the microarray slide only by the edges (do not touch the front or back of the slide), pick up the cover slip with the DNA side of the slide down (the label side is the DNA side) and quickly invert the slide and place in the chamber with the DNA side and cover slip up.

Pipet 11 μL of nuclease-free water into the wells in the hybridization chamber on either side of the slide. Place the cover on the chamber and lock the clips in place.

Working quickly and keeping the hybridization chamber horizontal, place the chamber at the bottom of the 37°C water bath. Incubate for 15-16 hours.

Day 5

Post-hybridization washing and scanning

Remove the hybridization chamber from the water bath and dry the outside with a large kim wipe. Pull the clamps off and wipe again before opening the chamber. Lift the slide from the barcode end using forceps. Remove the coverslip by quickly, but gently dipping the slide in a 50 mL conical tube filled with room temperature 1X SSC. Let the coverslip slide off gently; hold the slide at the barcode end with forceps. Once the cover slip comes off, transfer the slide to another conical tube filled with 50°C 1X SSC/0.1% SDS. Incubate for 15 minutes in the 50°C waterbath.

Transfer slide quickly to a second tube filled with 50°C 1X SSC/0.1% SDS. Do not let the slide dry out while transferring. Incubate for 15 minutes in the 50°C waterbath.

Transfer slide quickly to a third tube filled with 50°C 1X SSC/0.1% SDS. Do not let the slide dry out while transferring. Incubate for 15 minutes in the 50°C waterbath.

After the three washes are complete, rinse the slide in a tube filled with room temperature 1X SSC by plunging up and down 4-6 times.

Rinse the slide in a tube filled with room temperature 0.1X SSC by plunging up and down 4-6 times. When you pull the slide out the last time, pull it out very slowly so that water droplets do not form on the surface of the slide. Then carefully blot the edges of the slide dry on a folded kim wipe, being careful not to touch the front or back surface of the slide. Use the compressed air duster to blow off any water droplets that remain. You are not blowing the water dry, but chasing it to the edge of the slide so that it can be blotted by the kim wipe.

Carry the dry slide in a slide box covered in foil to the fume hood in the Keck Lab. Dip the slide into the coplin jar filled with 2% PEG (2000) in 1:1 acetone:toluene. Be careful not to dip the label into the solvent. Work quickly. Allow the slide to dry for about 1 minute before putting it back in the box and bringing it back into the Dahlquist lab for scanning. Keep the slide in the dark until scanning.

Scanning with the Axon GenePix 4000B Scanner

Make sure that the computer is OFF before turning on the scanner.

Remove the dust cover and turn on the scanner using the switch on the black box in the back of the scanner.

Turn on the computer and login to the username “scanner”. Ask Dr. Dahlquist for the password.

Let the scanner warm up for 15 minutes before scanning.

See the pictures on the cabinet above the scanner for how to insert the slide. Be gentle!

The DNA side of the chip should be down and the barcode should be facing towards the front of the instrument.

Open the Hardware Settings window (click the icon on the right-hand side that looks like the scanner with a key hanging off of it.)

Make sure that the pixel size is set to 10, lines to average is set to 2, and focus position is set to 0. Set the PMT Gain for the 635 wavelength to 650 and the PMT Gain for the 532 wavelength to 550. The Power should be at 100% for both.

Perform a Preview Scan by clicking the icon with the double arrows (looks like a "fastforward" icon).

While it is scanning, click on the histogram tab to see the Count Ratio of red to green signal. It should be within 10% of 1.00 (0.90 to 1.10). You can adjust the PMT Gain settings in the Hardware Settings window to adjust this ratio. The histogram tab will show you the data for the area of the image that is selected in the main Image window, so if you change the settings, you need to zoom to the area in which that setting applies.

Allow the Preview Scan to complete, so that the instrument will read the barcode. If it does not register the bar code, double-click on the barcode area at the bottom of the screen to enter it manually.

Perform a Data Scan by clicking on the single arrow (second icon down on the right-hand side of the window). It will take ~10 minutes to complete the scanning of the slide.

Save the data by clicking on the floppy/folder icon on the right hand side and choosing "Save Images" from the menu that appears.

In the dialog box that appears, make sure that the images are being saved as "Single-image tiffs". Use the barcode and date as part of the file names. Create a folder for today's date (yyyymmdd) in the C:\MicroarrayData folder to save the files.

When you are finished scanning, turn off the scanner so that we do not wear out the laser and replace the dust cover.

Notes

Please feel free to post comments, questions, or improvements to this protocol on the talk page. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.